PROJECT SUMMARY/ABSTRACT Mucosal tissues are frontline barriers that are continuously exposed to infectious pathogens. Developing and maintaining protective mucosal tissue-resident memory T cells (TRM cells) is necessary for immune surveillance. However, the mechanisms controlling homing and maintenance of mucosal TRM cells are unknown and likely distinct from those regulating conventional circulating effector memory (TEM) and central memory (TCM) cells. A currently accepted paradigm is that mucosal chemokines (i.e., CCL25, CCL28, CXCL14, and CXCL17) play a crucial role in mobilizing TRM cells in mucosal tissues, and may occur independent of local antigen recognition. However, several major gaps remain, including: (1) the identity of the mucosal chemokines involved; (2) their cellular source; and (3) the cellular and molecular mechanisms through which they regulate mobilization of anti- viral mucosal CD8+ TRM cells. This proposal uses herpes simplex virus type 2 (HSV-2) as a model viral pathogen to elucidate the underlying mechanisms that regulate mobilization of HSV-specific CD8+ TRM cells in the vaginal mucosal tissue. Our published and preliminary data indicate that: (A) Elevated numbers of HSV-specific memory CD62LlowCCR7lowCD103highCD8+ TRM cells and CXCR8+CD8+ T cells are present in the vaginal mucosa of HSV- 2 infected asymptomatic mice as compared to symptomatic mice; (B) Mucosal chemokines CXCL17 and CCL28 are highly expressed in the vaginal mucosal tissues of HSV-2-infected asymptomatic mice, with no apparent genital herpes disease; (C) CD8+ TRM cells that reside in vaginal mucosa of asymptomatic mice express CXCR3 (the receptor for T cell-attracting chemokines CXCL9 and CXCL10),CXCR8, and CCR10 (the receptors for CXCL17 and CCL28, respectively); (D) Intravaginal administration of T cell-attracting chemokines CXCL9 and CXCL10 is associated with increased recruitment of CXCR8+CD8+, CCR10+CD8+ TRM cells to the vaginal mucosa of asymptomatic mice; and (E) Exogenous CXCL9 and CXCL10 significantly increased expression of CXCR3, CXCR8, and CCR10 receptors on vaginal mucosa-resident CD8+ TRM cells. Based on these findings, we hypothesize that CXCL17 and CCL28 play a crucial role in homing and maintenance of vaginal mucosal tissue- resident CD8+ TRM cells, which protect from genital herpes. Our Specific Aims include: Aim 1 will identify the cellular sources of CXCL17 and CCL28 in vaginal mucosa and the mediators that regulate their expression. Aim 2 will investigate the role of CXCL17 and CCL28 mucosal chemokines and study the need for local viral antigen expression in developing, maintaining, and expanding CD8+ TRM cells within the vaginal mucosa. Aim 3 will develop a novel ?prime, pull, and keep? vaccine strategy to increase the size and maintenance of vaginal mucosa- resident CD8+ TRM cells to protect against genital herpes. This is based on three sequential steps of intravaginal administration of: (1) CD8+ T cell epitopes (prime); (2) CXCL9 and/or CXCL10 T-cell attracting chemokines (pull); and (3) CXCL17 and/or CCL28 mucosal chemokines (keep). Successful completion of this basic and translational research project will pave the way towards the development of an effective mucosal herpes vaccine.